Stimulation of interleukin-2 (IL-2) producing T cell clones with antigen and MHC class II molecules (Ia molecules) in planar lipid membranes or on chemically-modified antigen-presenting cells (APC) results in a state of long lasting proliferative nonresponsiveness to subsequent antigen stimulation in the presence of normal APC. Our recent experiments have shown that the induction of this take state takes place when T cell receptor occupancy occurs in the absence of a critical costimulatory signal delivered by the APC. When this signal is provided in the form of allogeneic APC, it prevents the induction of nonresponsiveness and stimulates the T cell to proliferate. A biochemical analysis of second messenger signals inside the cell revealed that the costimulatory signal does not pass through protein kinase c activation nor through the calcium-dependent calmodulin-mediated pathway, both of which are stimulated by T cell receptor occupancy. These results suggest that the T cell requires stimulation via a third biochemical pathway in order to make a full proliferative response. Lack of the third signal results instead in the induction of the nonresponsive state.